Thesis (M.Sc.)--York University, 2006. Graduate Programme in Kinesiology and Health Science. / Typescript. Includes bibliographical references (leaves 75-112). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://proquest.umi.com/pqdweb?index=2&did=1324366891&SrchMode=1&sid=4&Fmt=2&VInst=PROD&VType=PQD&RQT=309&VName=PQD&TS=1194985810&clientId=5220

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu). / This investigation was initiated to test the hypothesis that streptozotocin (STZ) and N-methylnitrosourea (MNU) damage B-cells of islet of Langerhans by depleting islets pyridine nucleotide content. Islets of Langerhans isolated by the collagenase method from nonnal rats were exposed to STZ rmder various experimental conditions and islets pyridine nucleotide content was temporally correlated with cytotoxicity. Metabolic and insulin secretory function of islets of Langerhans were used as indices of B-cell toxicity. The nitrosoureas (STZ and MNU) exerted time-, dose-, and temperature-dependent suppression of glucose-stimulated insulin secretion. Identical studies indicated that rmlike the nitrosoureas, alloxan was B-cytotoxic at 0°c even in the presence of 16.7 mM glucose. Whereas 2 mM alloxan elicited transient but consistent release of insulin by islets perifused in low glucose (1.7 mM) at 37°c, there was no release of insulin by islets perifused with 5 mM STZ, The effect of 5 mM STZ or of 10 mM MNU can be completely inhibited by the simultaneous presence of nicotinamide, isonicotinamide, picolinamide and other primary amides (e.g. pyrazinamide and benzamide) at concentration of 20 mM. Glucose (16.7 mM), pyridine nucleotides (10 mM) and acid derivative of amides (e.g. nicotinic acid; 20 mM) were ineffective. 2-Deoxyglucose (20 mM) or 3-0-methylglucose (20 mM) offered partial protection. After exposure of islets to STZ (5 mM) or MNU (10 mM), complete reversal of the effect was obtained with nicotinamide (20 mM) or its isomers, with time of exposure not exceeding 30 minutes. Beyond 30 minutes, the reversibility was partial or absent, except in the presence of 0.5 mM phenazine methosulfate (PMS) which induced release of insulin in both normal and nitrosoureas pre-treated islets. Insulin releasing action of PMS was dose-, time- and temperature-related; occurred even in the absence of glucose; was inhihitcd hy epinephrine (10 mM, hut not by mannoheptulose (20 mM); and was not potentiatcd by cyclic AMP (5 mM) or theophylline (10 mM). In the perifusion system, the patterns of response induced by PMS (0,5 mM) was spike-like release reaching a maximum in 5 minutes and declining rapidly to half-maximal value in 10 minutes. Normal islets pre-exposed to PMS was refractory to subsequent glucose (16.7 mM) stimulation. Islets pre-treated with STZ (1-5 mM) metaholized less 14c-glucose than control islets. The order of inhibition hy STZ of 14c-glucose metabolism by islet was: l-14C->U- 14C->6-14C-glucose. PMS (0.5 mM) augmented the metabolism of U-14C- and 1-14C glucose by STZ pre-treated islets. However, the metabolism of 6-14C-glucose was unelevated by PMS. The level of NADP+ + NADPII but not the level of NAD, decreased after two minutes exposure of islets to STZ. At thirty minutes, however, the levels of NAD,6-phosphogluconate and NADP+ + NADPII were decreased. The depletion paralleled suppression of insulin secretion. The level of NADP+ + NADPH in islets was decreased more than the level of NAD. Whereas PMS (0.5 mM) elevated the level of NADP+ + NADPH, the level of NAD was not augmented. Islets isolated from rats 3 or more hours after pre-injection with STZ (65 mg/kg) or 6-aminonicotinamide (40 mg/kg) failed to secrete insulin in response to glucose stimulation. However, insulin secretion by such islets was elevated in the presence of PMS (0.5 mM). Whereas PMS induced insulin secretion was much reduced 48 hours after preinjection of rats with STZ, the secretory activity in islets of rats 48 hours after 6-aminonicotinamide injection was slightly decreased. Pyridine nucleotides augmented secretion only in islets of 6-aminonicotinamide pre-injected rats. It is concluded that the immediate response of islets to nitrosoureas in vitro differs from islets response to alloxan. The actions of STZ and MNU are qualitatively similar. The B-cytotoxic effect of nitrosoureas is exerted directly or indirectly on the metabolic and energy coupling functions of pyridine coenzymes. Such an effect could be reversed by supplying the islets with reactive proton donors as substitutes for pyridine nucleotides.

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Made available in DSpace on 2014-06-11T19:29:52Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-20Bitstream added on 2014-06-13T18:39:58Z : No. of bitstreams: 1 spada_apm_me_botfm.pdf: 214670 bytes, checksum: 608905a90c46918e2a91642487aaed16 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Objetivo: avaliar o estresse oxidativo no sangue e na placenta de ratas com diabete de intensidade moderada. Métodos: O diabete foi induzido em ratas Wistar recém-nascidas (grupo diabete moderado) no dia do nascimento (dia 0) por streptozotocin (100 mg/kg, via subcutênea). As ratas do grupo nãodiabético (controle) receberam somente tampão citrato. Na vida adulta, as ratas (diabéticas e controle) foram submetidas ao acasalamento e o dia de diagnóstico positivo de prenhez foi considerado dia 0. A glicemia foi determinada nos dias 0, 7, 14 e 21 de prenhez. No 21º dia de prenhez, as ratas foram anestesiadas e dessangradas para determinação das atividades enzimáticas de superóxido dismutase (SOD), glutationa peroxidase (GSH-Px) e glutationa redutase (GSH-Rd) e das concentrações de grupos tiólicos (SH) e de espécies reativas ao ácido tiobarbitúrico (TBARS). Em seguida, as placentas foram retiradas e processadas para determinação das atividades de SOD e catalase e concentração de TBARS, gluationa reduzida e grupos tiólicos. Resultados: Ratas com diabete induzido no período neonatal (grupo diabético) apresentaram glicemia superior a 120mg/dl no dia 0 de prenhez e foi observada hiperglicemia no 14º dia de prenhez. A análise do estresse oxidativo em hemáceas lavadas mostrou que no grupo diabético houve aumento significativo na atividade da GSH-Px. No tecido placentário a atividade da catalase foi significativamente maior em ratas com diabete moderado. Conclusão: Frente às condições experimentais analisadas, o aumento dos biomarcadores do sistema antioxidante em ratas com diabete de intensidade moderada foram suficientes para conter o estresse oxidativo. / Objective: To evaluate the oxidative stress in blood sample and placental of female rats that received streptozotocin in the neonatal period. Methods: The diabetes was induced in female offspring (diabetic group) in the day of the birth (day 0) for streptozotocin (100 mg/kg, subcutaneous route). Female control rat (control group) received only citrate buffer. In the adult life, the female rats were submitted to the mating and the day the positive diagnosis, was considered day 0 of pregnancy. The glycemia was measured in the 0, 7, 14 and 21 of pregnancy. At day 21 of pregnancy, the female rats were anesthetized and died by decapitation for collection of the blood for determination of the enzymatic activity of the superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and glutathione reductase (GSH-Rd) and of the concentrations of thiols group and thiobarbituric acid reactive substances (TBARS). Afterwards, placental were removed and processed for determinations of the enzymatic activity of the SOD and catalase and of the concentrations of the TBARS, glutathione reduced (GSH) and thiols group (SH). Results: Diabetic rats presented blood glucose concentration greater than 120 mg/dL in the day 0 of pregnancy and hyperglycemia in 14 º day of pregnancy. The analysis of the oxidative stress in maternal blood sample showed increased in GSH-Px activity. In placental tissue catalase activity of diabetic group is found to be increase in homogenate tissue in diabetic group. Conclusion: The hyperglycemia in diabetic rats increased antioxidant system biomarkers, however, these alterations were enough to control oxidative stress.

We have examined the effect of recent onset diabetes on several aspects of hepatic microsomal metabolism in both chemically-induced and spontaneously BB (Bio Breeding) diabetic male and female Wistar rats. Experiments were performed either 4 days post-streptozotocin injection or 4 days after withdrawal of insulin (BB rats). Differential alterations of the diabetic state on hepatic microsomal enzyme activities were observed. Female diabetic rats exhibited no change in benzo[a]pyrene hydroxylase activity, a decrease in testosterone A⁴ hydrogenase, and an increase in aniline hydroxylase. On the other hand, male diabetic rats demonstrated a decrease in hepatic benzo[a]pyrene hydroxylase activity, no change in testosterone A⁴ hydrogenase, and an increase in aniline hydroxylase. Insulin treatment reversed these effects. Benzo[a]pyrene hydroxylase kinetic studies did not reveal marked differences between control and diabetic rats. There were no marked differences between the chemically-induced and genetic models of diabetes with respect to the metabolism studies. Serum testosterone levels were significantly lower than control in BB diabetic males, whereas no change was apparent in female diabetics. Serum insulin determinations suggested that the BB diabetic animals we examined were not severely diabetic although they did exhibit hyperglycemia. Electrophoresis of hepatic microsomal proteins indicated that spontaneous diabetes of short duration altered the protein distribution in the cytochrome R450 region. Two [³H]-estradiol binding sites were detected in rat liver cytosol by Scatchard analysis with a ligand concentration range of 0.05 to 200 nM. The high affinity site, which was specific for estrogens, exhibited a K[sub=d] of ~10⁻¹⁰M and a capacity of ~100 fmol/mg protein in the 50% ammonium sulfate fraction. Unexpectedly, the data suggested that the capacity of this site was greater in males than in females. The moderate affinity binding site exhibited a k[sub=d] of ~10⁻⁷M and a capacity of M0 pmol/mg protein in the whole cytosol fraction. Binding at this site was markedly pH dependent. Both estradiol and dihydrotestosterone competed for binding to this site. A sex difference existed for moderate affinity binding because it was present only in males. We obtained unexpected results in binding studies conducted on a relatively small number of BB diabetic rats. In diabetic males, the capacity of the high affinity site was reduced to 50% of control, whereas the reduction in moderate affinity binding was not nearly so marked. Additional studies using a larger sample size and more sophisticated data analysis are required to verify these results. We concluded that alterations in sex dependent drug metabolism evident in streptozotocin-induced diabetic rats were also seen in the spontaneously diabetic rat model, and were accompanied by changes in the relative disposition of electrophoretically separable microsomal proteins. Changes in circulating androgen levels were also found in BB diabetic males, along with changes in the capacities of certain hepatic steroid binding sites. It is not yet possible to establish mechanistic relationships between these [³H]-estradiol binding sites and modulation of hepatic drug metabolism. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

Indiana University-Purdue University Indianapolis (IUPUI) / Bone disease and degradation is a ubiquitous problem, the complexity and treatment of which humanity has only begun to understand. Diabetes Mellitus is a disease which, in all forms, profoundly effects the organs of the body, bone included. As is often the case in biology, there are inherent differences between the sexes when considering skeletal development and disease progression and outcome. Although there are several reported mouse models for diabetes, until now there has been no characterization of bone disease in any model where diabetes occurs with equal frequency in males and females in greater than 90% of animals. In this study, a protocol for reliable induction of diabetes in both sexes using intraperitoneal injections of Streptozotocin was developed. The resulting bone phenotype in male and female mice was characterized and compared to weight and age matched control groups. In this model female diabetic mice exhibited a robust deficit in bone quality, while both sexes experienced loss of beta-cell mass and increased glycation of hemoglobin rendering the diabetic mice unable to produce insulin endogenously. Further, these mice were unable to metabolize exogenous insulin injected during insulin tolerance testing. This model is a strong candidate for future exploration of osteoporotic bone disease, Diabetes Mellitus, and the link between estrogen and glucose sensitivity.

Yes / Medical Research Council, Innovate UK and Epistem Ltd.

The increasing reliance on drugs from natural sources has led to the development of several drugs from traditional plants which are present in abundance in Southern Africa. With the rapid increase of incidence of type 2 diabetes in South Africa with potentially devastating effects on healthcare, the need for alternative therapeutics is a priority. In this study, Schkuhria pinnata (Lam.) Kuntze was investigated for its antidiabetic potential. Initial screening of two different solvent extracts of S. pinnata identified an aqueous extract that lowered blood glucose concentrations in a hyperglycaemic streptozotocin-induced diabetic rat. The classical bioassay approach was followed by using different solvents, drying processes and fractionation in order to produce the most active extract and attempt to isolate an active compound(s). An aqueous freeze dried extract was found to be the most active at stimulating glucose uptake activity in C2C12 and Chang cells. Fractionation of this extract in an attempt to identify the active compound yielded a novel crystalline compound 1 by NMR analysis. Screening for bioactivity of the extract and compound 1 using C2C12 muscle and Chang cells revealed that both extract and compound 1 were biologically active, however the activity of the aqueous extract was more significant overall. A butanone/pentane extract prepared for possible commercialization purposes was also shown to be active in vitro. To establish antidiabetic activity, the aqueous freeze dried extract, butanone/pentane extract and the enriched compound 1 fraction were tested in a streptozotocin (STZ) diabetic rat model showing hypoglycaemic effects for the aqueous freeze dried extract. Messenger RNA and protein studies on C2C12 muscle cells revealed that the aqueous freeze dried extract and compound 1 enhanced insulin receptor, GLUT-4, glycogen synthase, pyruvate kinase and pyruvate carboxylase expression, suggestive of an insulin mimetic mode of action, while the butanone/pentane extract enhanced adenosine monophosphate-activated kinase (AMPK) protein expression by a non-insulin dependent mechanism. A pharmacokinetic study (PK) established bioavailability of compound 1 following oral administration of the extracts, but not from the compound 1 enriched fraction. From this study, the traditional use of S. pinnata has been scientifically validated as having antidiabetic properties. In vitro and in vivo bioassays, confirmed that an aqueous freeze dried extract which was prepared as per the traditional method had the most promising antidiabetic iii activity. Compound 1 isolated from an active fraction was proven to be almost as effective as the parent extract in in vitro studies. This compound could therefore be the major active ingredient responsible for the uptake of glucose in cells and the hypoglycaemic activity in vivo. In this study, the antidiabetic activities together with the mechanism of action of S. pinnata extracts and compound 1 were elucidated. The highlight of the study was the identification of a bioactive novel chemical entity (NCE) compound 1 (identified as 2-(2-{[(2E)-4-hydroxy2-(hydroxymethyl)but-2-enoyl]oxy}-4,7-dimethyl-1,2,3,4-tetrahydronaphthalen-1-yl)prop-2- enoic acid) isolated from an active fraction of S. pinnata that was proven to be almost as effective as the parent extract in in vitro studies. This compound could therefore be the major active ingredient responsible for the uptake of glucose in cells and the hypoglycaemic activity in vivo. The cellular mechanism of action of the S. pinnata extracts and compound 1 demonstrated both insulin mimetic and non-insulin dependent mechanisms (AMPK) in C2C12 muscle cells. Further research in the form of preclinical and clinical trials need to be undertaken to make this extract or biologically active compound available as a herbal remedy or nutraceutical therapeutic for diabetes. To achieve this; safety, efficacy and mode of action studies will have to be established. The synthesis of compound 1 and/or analogues should also be investigated as an antidiabetic drug candidate.

Diabetic cystopathy is a chronic and common complication of diabetes with a classical triad of symptoms; decreased bladder sensation, increased bladder capacity and impaired detrusor muscle contractility (Hunter and Moore, 2003). In animal models of diabetes such as streptozotocin-induced diabetes in the rat, abnormalities of bladder function have been reported (Longhurst and Belis, 1986). The prototypic TRPV channel, TRPV1, is activated by capsaicin, which has been shown to cause contraction of the rat bladder (Saitoh et al., 2007), and this is reduced in STZ-diabetic rat bladder (Pinna et al., 1994). Therefore we hypothesize that TRPV1 function will be reduced in the diabetic bladder. The aim of this study are the following: Firstly, to investigate the effect of the streptozotocin (STZ) model of diabetes on a range of TRP channel functions in the urinary bladder smooth muscle preparation using TRP channel agonists and antagonists and to study the neurotransmitters involved in the contractile or relaxant responses. Some studies were also performed on colon tissues. Secondly, to explore the involvement of cholesterol modudation in TRP channel signalling. Thirdly, to study the change in TRP channel response with time following the treatment with streptozotocin. The results showed that the contractile responses to the TRPV1 agonist capsaicin, TRPV4 agonist 4-α-PDD, and TRPA1 agonist allyl isothiocyanate were significantly reduced in diabetic bladder. The selective TRPV1 antagonist, SB-366791, inhibited the contractile responses to capsaicin confirming the involvement of TRPV1 channels. The effect of diabetes is unlikely to be at the level of contractile machinery since the contractile responses to muscarinic receptor agonist carbachol were not significantly reduced in diabetic tissues. It is reported for the first time that the combination of neurokinin 1 and 2 antagonists GR-205171 and SB-207164 inhibited the contractile responses to capsaicin suggesting that a neurokinin may be the neurotransmitter involved in the capsaicin responses. In addition, the reduction of the responses to capsaicin in STZ-induced diabetic tissues occurred not only in urinary bladder but also in colon. Cholesterol-PEG significantly lowered the maximal contractile responses to capsaicin of rat bladder strips. Methyl-β-cyclodextrin, α-cyclodextrin and β-cyclodextrin at the same concentrations enhanced the contractile responses to capsaicin in the control and diabetic rat bladder strips. These effects of cyclodextrin are specific to capsaicin activated contractions and not seen with TRPA1 activation, suggesting that the effects are not mediated downstream of channel activation. Since α-cyclodextrin does not sequester cholesterol, the enhanced responses to cyclodextrins may not be due to the cholesterol modulations. Instead, theses novel findings may possibly occur by changing the local membrane lipid environment of the TRPV1 channel. As early as 36 hours after induction of diabetes by STZ, the contractile responses to capsaicin were significantly reduced in comparison to those of the controls and this reduction persisted until the eight weeks time point. In contrast, responses to the TRPA1 agonist allyl isothiocyanate were not affected at early time points but were reduced one week after STZ treatment. This detailed time course analysis suggests that there are novel mechanisms of modulation of the TRPV1 channels in this STZ model. In conclusion, in the rat urinary bladder or colon preparations, diabetes mellitus using STZ animal model caused 1) the impairment of a number of TRP channel subfamily functions, TRPV1, TRPV4 and TRPA1 but not TRPM8. The combination of NK1 and NK2 antagonists significantly inhibited the responses to capsaicin. This may suggest the involvement of neurokinin in postsynaptic transmission in rat bladder following the activation of TRPV1 channel, 2) the impairment caused by STZ-induced diabetes occurred very early (within 36 hours after diabetes induction) in TRPV1 channel but not TRPA1 channel. There are specific early effects of STZ treatment on TRPV1 channel function at a time when other afferent nerve terminal channels (TRPA1) are functioning normally, suggesting that early onset of dysfunction in TRPV1 signalling may not merely be the consequence of nerve damage, 3) the mechanism of this impairment may not be the effect of neuropathy on neurotransmitter release or nerve damage. Improving the responsiveness of nerves of bladder in diabetic patients might be of therapeutic benefit. The present studies suggest that it is possible to enhance function using indirect modulators such as bradykinin which potentiated the TRPV1 channel function in diabetic rat bladders.